Water is the medium of life. Since biological membranes have only limited intrinsic water permeability cells maintain the flux of water into and out of the cell via a family of water-specific, membrane protein channels called aquaporins (1). Members of the aquaporin family are found in archea, eubacteria and eukaryotes, including fungi, animals and plants. They serve an astonishing variety of physiological functions (5-7) and are easily identified by sequence similarity across all kingdoms of life. In higher eukaryotes, water transport activity of aquaporins is frequently regulated by phosphorylation, pH and osmolarity (6-8). Aquaporins in plants and animals are highly conserved and form large protein families with 35 members in higher plants (9) and 13 members in humans (5,10).
Based upon phylogenetic analyses, plant aquaporins are further divided into four subfamilies and their presence in primitive plants such as the bryophyte Physcomitrella patens implies that this specialization was already present in an ancient plant-ancestor (11). There are 13 remarkably conserved plasma membrane aquaporins (Plasma membrane Intrinsic Proteins or PIPs) which are all regulated, and these further separate into two distinct phylogenetic groups (PIP1 and PIP2).
Closure of the plant aquaporin SoPIP2;1 of spinach (formerly called PM28A (2)) has been reported to be triggered by the dephosphorylation of two serine residues: Ser115 in the cytosolic loop B (conserved as Ser in 12, and as Thr in 1, of the 13 Arabidopsis PIPs) and Ser274 in the C-terminal region (2,3) (conserved as Ser in 7, and as Thr in 1, of the 8 Arabidopsis PIP2s). Both residues are situated in consensus phosphorylation sites. Furthermore, the simultaneous closure of all Arabidopsis PIPs upon anoxia was recently reported to depend upon the protonation of a strictly conserved histidine residue in loop D (4), which corresponds to His193 in SoPIP2;1 (SEQ ID NO: 33). It is an intriguing observation that distinct chemical signals acting on residues well separated in sequence induces an identical physiological response within PIPs. While a number of structures have been reported for water (12-16) and glycerol (17) channels, no plant aquaporin structure has yet been determined at high resolution. Gonen et al. (15) reports a low-resolution structure of AQP0. At this resolution (3 Å) water molecules cannot be seen and the authors are not able to conclude that the structure represents a closed aquaporin. This is also clearly stated by the authors in the article (p 194-195: “We note, however, that our resolution is currently limited to 3 Å, and even if a pore appears to be in a closed conformation, it might still be permeable to solutes.”). Furthermore, a high-resolution structure of AQP0 (16) with an open conformation show no global change in the structure as compared to the low-resolution AQP0 structure reported in ref 15. Thus, it is likely that the structure in ref 15 represents an open aquaporin. Recently an additional report (35) arrives at the same conclusion that the structure of AQP0 reported in ref 15, as well as in ref 16, is open and not closed to water transport.
In addition, the low-resolution structure of AQP0 presented in Gonen et al. (15) is based on a proteolytically cleaved AQP0. Thus, both the N- and C-terminal regions of the protein are cleaved off and can therefore not participate in closing the pore (36). Kukulski et al. (37) depicts a 5 Å low-resolution structure of an aquaporin that in a previous publication by the same authors had been shown to be open (28). However, from a 5 Å low-resolution structure it is impossible to see if the pore is open or closed.
Furthermore, no gating mechanisms have been unambiguously demonstrated. Therefore it is crucial to establish the atomic structure of an aquaporin in its closed formation. Structural information of the closed conformation is necessary for understanding the mechanism of gating and for structure-based design and development of organic compounds, peptides or antibodies that either stabilize the open conformation or the closed conformation. By obtaining the structure of a closed aquaporin it will for the first time be possible to use that particular structure to modify the gating. This can in plants be done by direct genetic engineering of aquaporins in order to improve stress tolerance, e.g. against drought stress. In mammalian species pharmaceutical compounds that stabilize the closed or the open conformation of aquaporins can be designed based on the closed conformation of the aquaporin SoPIP2;1 (SEQ ID NO: 33) from the plasma membrane of the plant spinach. Such inhibitors and activators are candidate pharmaceutical and cosmeceutical compounds, e.g. antiperspirants. Aquaporins are also important for cell migration during angiogenesis, wound healing, tumour spread and organ regeneration (27), processes that therefore can be modulated by pharmaceutical compounds interacting and modifying the gating of aquaporins. Dysfunction of human aquaporins is associated with clinically important diseases such as polyuria in kidney diseases. Conversely, increased water retention is associated with congestive heart failure, liver cirrhosis and nephritic syndrome. Also pathological skin conditions such as anhidrosis, hyperhidrosis and conditions where the transepidermal water loss is deviating from normal conditions could be targets for aquaporin inhibitors and activators. Moreover, brain edema, glaucoma and skin burns could be treated by inhibitors and activators of aquaporins. The cosmeceutical applications of aquaporin inhibitors and activators include not only antiperspirant but also dermatological conditions where one wants to influence the transepidermal water loss. The atomic structure of the closed conformation of SoPIP2;1 (SEQ ID NO: 33) can also be used for designing novel in silico and in vitro screening systems for pharmaceuticals and cosmeceuticals acting as modulators of aquaporin gating and function. Knowledge of the atomic structure of the closed conformation can also be used to design, and also to screen for, peptides and antibodies that interact with certain epitopes on aquaporins and thus effect activity and gating.